Post protein hydrolysis removal of a potent ribonuclease inhibitor and the enzymatic capture of DNA

ABSTRACT

The present invention concerns compositions and methods of extracting infectious pathogens from a volume of blood. In one embodiment, the method includes the steps of creating a fibrin aggregate confining the pathogens and introducing a fibrin lysis reagent to expose the pathogens for analysis. The present invention also concerns materials and methods for removing aurintricarboxylic acid (ATA) from a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/117,505, filed May 8, 2008; which is a divisional application of U.S.application Ser. No. 11/035,667, filed Jan. 14, 2005; U.S. applicationSer. No. 11/035,667 is a continuation-in-part of U.S. application Ser.No. 10/604,779, filed Aug. 15, 2003; U.S. application Ser. No.10/604,779 claims the benefit of U.S. Provisional application Ser. No.60/319,474, filed Aug. 15, 2002, and U.S. Provisional application Ser.No. 60/319,803, filed Dec. 19, 2002; U.S. application Ser. No.11/035,667 claims the benefit of U.S. Provisional application Ser. No.60/481,892, filed Jan. 14, 2004; U.S. application Ser. No. 11/035,667 isa continuation-in-part of International application No.PCT/US2004/026606, filed Aug. 16, 2004; which claims priority to U.S.application Ser. No. 10/604,779, filed Aug. 15, 2003, and claims thebenefit of U.S. Provisional application Ser. No. 60/481,892, filed Jan.14, 2004; the disclosure of all of which is incorporated herein byreference in its entirety.

This invention was made with government support under Grant No.DAAD13-01-C-0043 awarded by the U.S. Army Soldier and BiologicalChemical Command. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The threat of bioterrorism (BT) and biological warfare presentschallenges for the clinical setting that are best met with rapid andsensitive technologies to detect BT agents. Peripheral blood samplescould contribute to early and specific clinical and epidemiologicalmanagement of a biological attack if detection could take place when theconcentration of the infecting organism is still very low. The worriedwell and recently infected patients would benefit, both psychologicallyand physically, from early pharmacological intervention.

Infection with Bacillus anthracis or Yersinia pestis often presentinitially as a nonspecific febrile or flu-like illness. Themediastinitis associated with inhalational anthrax ultimately results inbacilli entering the blood once the efferent lymphatics become ladenwith organisms. When bacteremia (the presence of bacteria in the blood)and sepsis (the invasion of bodily tissue by pathogenic bacteria) haveinitiated, the number of bacilli may increase quickly, doubling every 48minutes, most often resulting in death of the patient.

It has been reported that microbiological studies on patient bloodsamples are useful for diagnosing pneumonic plague. The potential forYersinia pestis bacilli to be present in peripheral circulating bloodsuggests that a PCR assay would make a useful diagnostic tool. Testingfor pneumonic plague or inhalational anthrax would be effective whenhealthy patients present with “flu-like” symptoms (malaise, fever,cough, chest pain and shortness of breath) that may accompany othernonspecific symptoms. However, in order to maximize the probability ofsuccessful treatment, detection of the infecting organism must takeplace early in the disease process, when the concentration ofcirculating bacteria is very low.

Extraction of pathogen DNA from whole blood typically requires between200 μl to 500 μl of whole peripheral blood patient sample for eachpreparation event. Detection of early bacteremia is improved by using anentire 6 to 10 ml tube of patient blood for a single sample preparationevent. Prior art literature describes a single tube blood culture systemexploiting the selective lysis of blood elements, followed bycentrifugation to pellet bacteria for plating on solid media. Thetechnique has been examined thoroughly in conjunction withmicrobiological testing. Previous methods based on lyses of blood cellsfollowed by centrifugation have not proven to be useful for nucleic acidor biosensor based detection protocols.

Accordingly, what is needed in the art is: 1) a method of destroying andmaking soluble the spectrum of blood element components (erythrocytes,leukocytes, nuclear membranes, fibrin, and host nucleic acid) withoutdamaging analyte particles (bacteria, virus, fungi, toxin, metabolicmarkers, disease state markers, or chemical agents) in order to exposeand rapidly concentrate (via centrifugation, filtration, or capture) theanalyte particles from large volumes of blood, 2) processing to minimizeinhibition and/or removal of the host DNA and the matrix associatedbiomass present in the large volume blood sample using a single stepenzyme detergent cocktail that is amenable to automation and portablesystems, and 3) an analyte particle concentration method that can becoupled to existing manual or automated processes for nucleic acidextraction, biosensor testing, or liquid chromatography separation andmass spectrometry analysis. It is, therefore, to the effectiveresolution of the aforementioned problems and shortcomings of the priorart that the present invention is directed.

Fibrin is an insoluble protein precipitated from blood that forms anetwork of fibers. In vivo, this process is central to blood clotting.Fibrin is created by the proteolytic cleavage of terminal peptides infibrinogen. In the laboratory analysis of blood, an aggregate (pellet)of fibrin combined with other blood elements sediments at the bottom ofa tube when blood is centrifuged. Within the fibrin aggregate, pathogensare trapped. The analysis of these pathogens is highly desirable.However, like coins embedded in a slab of concrete, the capturedpathogens are substantially hidden from analysis, trapped in the fibrinaggregate. For individuals potentially exposed to dangerous pathogens,time is of the essence and rapid identification of the capturedpathogens is paramount. Rapid identification of nucleic acid, proteins,or other molecules associated with bacteria, virus, fungi, toxin,metabolic markers, disease state markers, or chemical agents isimportant for individual clinical management as well as forensic andepidemiological investigation.

Plasmin is a substance in blood capable of converting fibrin tofibrinogen monomers. Plasminogen is a precursor of plasmin in the blood.Streptokinase is an enzyme that activates plasminogen to form plasmin.The combination of plasminogen and streptokinase in the presence of thefibrin aggregate containing blood elements and bacteria (formallypresent in peripheral circulation) allows the conversion of the fibrinaggregate to a liquid state. Plasminogen activators are naturallyoccurring enzymes found in most all vertebrate species. These enzymes inany combination can also be used to derive beneficial blood matrixdisassembly where the downstream application require clots or bloodelement aggregates to be dissolved in order to facilitate sample flowand analyte interrogation.

Aurintricarboxylic acid (ATA) is a polymeric anion that has beendemonstrated in the literature to be a potent ribonuclease inhibitor.The compound has been described previously as an additive to samplelysis buffers where the objective is to extract RNA species from tissuesamples. The nucleic acid extract derived from such procedures has beenshown to be suitable for hybridization and gel electrophoresis analysis.However, ATA is a potent inhibitor of reverse transcriptase, which isessential for the polymerase chain reaction (PCR) detection of RNAspecies. Published procedures to remove ATA from nucleic acid containingcompositions have revolved around chromatographic procedures thateliminate or remove only a portion of the ATA.

The use of ATA in a proteinase K lysis buffer is potentially superiorto 1) chaotrophic salts (since they tend to reduce the efficiency ofproteinase K driven protein hydrolysis as evidenced by PCR results); 2)protein based ribonuclease inhibitors (since these inhibitors would bebroken down by proteinase K); and 3) EDTA (which only indirectlyinhibits nucleases via chelation of the divalent cations used by thosenucleases). In fact, divalent cations must be added to RNA preparationswhere enzymatic DNA hydrolysis is conducted. What has not beendemonstrated in prior art is a method where, once added, the completedownstream removal of ATA from nucleic acid extracts can be achieved tothe point that downstream reverse transcriptase PCR (RT-PCR) willfunction. Also not previously described is a way to utilize ATA in alyses buffer to treat a large volume (1 to 10 ml) of whole blood sampleand after several reagents addition steps move directly to RNA arrayhybridization using the entire blood sample for one analysis event hencebypassing RNA extraction and amplification.

Also not previously described is a way to selectively allownon-diagnostic RNA species residing outside the nucleus of leukocytes tobe degraded by endogenous and or exogenous nucleases while diagnosticRNA which mostly resides inside the nucleus (RNA that for instanceindicates up or down regulation of genes) is preserved enough for arrayor amplification based detection. Typically, chemistries that do notprovide abundant intact ribosomal RNA are not further examined becauseend users skilled in the art use such non diagnostic RNA species tojudge overall RNA integrity. Based on biochemical and phenotypicaldifferences between phospholipid membranes found in various bloodelements and the combined biochemical activity characteristics of thereagent cocktail, RNA species such as globin and ribosomal RNA aredestroyed but the diagnostic mRNA which is used to detect presence orabsence of various disease and or pathological processes is preservedenough for identification. Also, by allowing for the bulk ofnon-diagnostic RNA to be destroyed, there is less inhibition of PCR(polymerase chain reaction) contributed by the nucleic acid extract.

ATA also serves an important function in the protection of bacterial DNAwhen that bacteria is present in a blood sample processed with reagentscontaining high levels (≧100 U/ml) of DNase I as is used in variousembodiments contained within U.S. application Ser. No. 10/604,779. Inorder to achieve RNA detection capabilities that are superior to whatcan be achieved with technology descried in U.S. application Ser. No.10/604,779, and to do so without additional steps or requirements, thepresent invention is utilized in combination with blood sample treatmenttechnology described in U.S. application Ser. No. 10/604,779 and priorart nucleic acid extraction methods that utilize chaotrophic salts suchas guanidine thiocyanate in the presence of capture matrices such assilica or methods that utilize precipitation methods to concentratenucleic acids out of crude samples.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns compositions and methods of extractingand detecting infectious pathogens from a volume of blood. In oneembodiment, the method includes the steps of creating a fibrin aggregateconfining the pathogens; introducing an enzyme based Blood ProcessingReagent to expose the pathogens for analysis and to facilitate pathogenDNA extraction. In one embodiment, the enzyme based Blood ProcessingReagent may be composed of DNAse, plasminogen and streptokinase frozenin coincident relation until the fibrin lysis reagent is needed wherebystreptokinase enzymatically reacts with plasminogen to form plasmin uponthawing and introduction into the fibrin aggregate sample. The DNAseenzyme is used to facilitate the chemical and physical disruption ofpelleted blood elements that result from the previously describedprotocol in addition to other benefits described herein. Preferably, theplasminogen is suspended in an aqueous salt solution, including NaCl andNa₃PO₄, prior to freezing. The fibrin lysis reagent can also comprisePhospholipase A₂. Phospholipase A₂ is used to help non-pathogen DNAdigestion by destroying phospholipid bilayers and, hence, destruction ofthe nuclear membrane.

The subject invention also concerns materials and methods forefficiently removing ATA from a nucleic acid composition. The subjectmethods provide a nucleic acid composition sufficiently free of ATA suchthat a RT-PCR reaction and other reactions involving reversetranscriptase can be performed.

The subject invention also concerns materials and methods for a mixtureof ATA, magnesium chloride, potassium phosphate, and sodium chloridethat is dried and combined with other dried components such as thosedescribed herein.

The subject invention also concerns materials and methods for heating asolution of urea, DTPA, optionally containing EDTA, sodium citrate, andsodium chloride, to between about 400 to 600° C. for 1 to 4 hoursfollowed by drying and combination with proteinase K and optionallyMethyl 6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside and the use of thissample Lyses Reagent to allow ATA removal from nucleic acid extractsmade with existing prior art methods based on chaotrophic salts ornucleic acid precipitation followed by centrifugation or methodsdescried herein to allow downstream hybridization of RNA speciesdirectly out of treated whole blood samples.

The subject invention also concerns the urea/DTPA sample Lyses Reagentthat was heat treated to 400 to 600° C. for 1 to 4 hours duringproduction and used in sample treatment as descried above followed bythe combination of urease to break down the urea followed by RNA arrayanalysis.

The subject invention also concerns materials and methods for pathogencapture using bioactive peptides functionalized on hyaluronic acid asdescribed herein where the hyaluronic acid in turn acts as a polymericwaveguide.

The subject invention also concerns a way to cause a calcium release atthe site of pathogen capture via bioactive peptide or annealing of RNAspecies so as to trigger the conversion of reporter molecule labeledfibrinogen to insoluble fibrin at the site of pathogen capture viabioactive peptide or annealing of RNA species upon the matrix of thehyaluronic acid polymeric waveguide.

The subject invention also concerns materials and methods where thehyaluronic acid matrix that is cross linked utilizing biotin andstrepavidin and functionalized with bioactive peptides, such as thosedescribed herein, can be subsequently broken down with hyaluronidase inorder to facilitate pathogen elution.

The subject invention is practiced in conjunction with methods andmaterials for extracting infectious pathogens from a volume of a sample,such as blood, including the steps of creating a fibrin aggregateconfining the pathogens and introducing an enzyme based Blood ProcessingReagent to expose the pathogens for analysis and DNAse to facilitate DNAextraction specified in U.S. application Ser. No. 10/604,779, filed Aug.15, 2003. The enzyme based Blood Processing Reagent may be composed ofDNAse, plasminogen and streptokinase frozen in coincident relation untilthe fibrin lysis reagent is needed whereby streptokinase enzymaticallyreacts with plasminogen to form plasmin upon thawing and introductioninto the fibrin sample. Preferably, the plasminogen is suspended in anaqueous salt solution prior to freezing including NaCl and Na₃PO₄. Theenzyme based Blood Processing Reagent is preferably composed of DNAseand Phospholipase A₂. The DNAse enzyme is used to facilitate thechemical and physical disruption of pelleted blood elements that resultfrom the previously described protocol. Phospholipase A₂ is used to helphuman DNA digestion by destroying phospholipid bilayers and, hence,destruction of the nuclear membrane.

The subject invention also pertains to materials and methods forefficiently removing human DNA that has been processed or cleaved byDNAse, endonuclease, or exonuclease while pathogen DNA remains insideintact pathogens. Single Strand Binding (SSB) proteins are known in theart to enhance PCR kinetics through binding to DNA and can also be usedin the methods of the invention. In an exemplified embodiment, the SSBis biotinylated and the solid matrix has avidin or streptavidin attachedto the surface, and the SSB is bound to the matrix via the biotin-avidinbinding. In one embodiment of the method, a purified nucleic acidextract sample is optionally combined with proline at 2 to 20 mM and orDTT at 2 to 5 mM then circulated for several minutes at about 37° C.with the immobilized SSB. The SSB-matrix and bound human DNA isseparated from the sample and the remaining sample collected. Theremaining sample contains nucleic acid with a reduced human DNA load andcan be used for PCR testing. Since DNAse, endonuclease, or exonucleasewill nick human DNA in the presence of ATA and or other nucleaseinhibitors described herein combined with the other biochemical elementsdescribed in U.S. application Ser. No. 10/604,779, while pathogen DNAresiding inside intact pathogen structures is not nicked, a portion ofthe inhibitory human DNA can be selectively removed in this way postnucleic acid extraction.

The subject invention also pertains to the use of nuclease inhibitorswith or without ATA plus high levels of DNAse, endonuclease, orexonuclease (over 200 U/ml). The combination of nuclease inhibitors andnucleases teaches against the art but leads to processing of human DNAso that said DNA presents a small inhibitory contribution to PCRreactions compared to the same amount of human DNA that is not contactedwith this reagent mixture.

In applications when RNA purification is desired, solutions used in thesubject methods should be RNase-free. RNase-free solutions can beprepared using methods known in the art, including treatment with DEPC,typically at about 0.1%. DEPC treated water should be used to wash andrinse any glass or plasticware used in RNA isolation methods that is notRNase-free. Residual DEPC should always be eliminated from solutions orglassware/plasticware by autoclaving or heating to 100° C. for 15minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view of the method according to the inventionaccording to the invention.

FIG. 2 is a diagrammatic view of the preparation of the fibrin lysisreagent according to Protocol 1 of the invention.

FIG. 3 is a diagrammatic view of the setup of extraction reagentsaccording to Protocol 1 of the invention.

FIGS. 4-5 are diagrammatic views of bacterial recovery and fibrin lysisaccording to Protocol 1 of the invention.

FIG. 6-9 are diagrammatic views of bacterial lysis and nucleic acidextraction according to Protocol 1 of the invention.

FIG. 10A is a diagrammatic view of the steps of extracting reagentsaccording to Protocol 2 of the invention.

FIG. 10B is a diagrammatic view of the steps of extracting reagentsaccording to Protocol 2 of the invention.

FIG. 11 is a diagrammatic view of the steps of extracting reagentsaccording to Protocol 3 of the invention.

FIG. 12A is a diagrammatic view of the steps of extracting reagentsaccording to Protocol 4 of the invention.

FIG. 12B is a diagrammatic view of the steps of extracting reagentsaccording to Protocol 4 of the invention.

FIG. 13 is a table providing data on noise band crossing points forblood samples spiked with B. anthracis and processed with plasminogen,streptokinase, phospholipase A₂, DNase I, and lipase with centrifugationor filtration.

FIG. 14 shows sedimentation and solublization of tissue aggregates from6 ml blood samples exposed to various detergent and enzyme treatments.

FIG. 15 shows filtration characteristics of 6 ml blood samples exposedto various detergent and enzyme treatments.

FIG. 16 shows the results pathogen detection by canonical SNP analysisusing the present invention.

FIG. 17 is a bar chart showing detection of CRP in whole blood treatedin a Blood Processing reagent of the present invention.

DETAILED DISCLOSURE OF THE INVENTION

The present invention concerns compositions and methods of extractingand detecting infectious pathogens, components thereof, or other matter,such as prions, toxins, metabolic markers, cancerous matter or markers,disease state markers, and the like, from a volume of blood or otherbiological sample from a patient, such as a human or other mammal. Inone embodiment, the method comprises preparing a fibrin aggregate of ablood sample to confine pathogens; contacting the fibrin aggregate witha fibrin lyses reagent to release pathogens or pathogen componentstrapped in the aggregate; lysing pathogens and/or extracting pathogennucleic acids or other pathogen components; and detecting the pathogens,nucleic acids and/or components. Pathogen analysis can be accomplishedby any appropriate means including, but not limited to, blood culture,antibody based testing, or nucleic acid sequence based testing (PCR,Reverse Transcription PCR, NASBA, TMA or the like).

In one embodiment, the fibrin lysis reagent may be composed of anuclease, plasminogen and streptokinase frozen in coincident relationuntil the fibrin lysis reagent is needed. Upon thawing and introductioninto the fibrin aggregate sample, streptokinase enzymatically reactswith plasminogen to form plasmin. The nuclease enzyme facilitates thechemical and physical disruption of pelleted blood elements. Nucleasescontemplated within the scope of the present invention include, but arenot limited to, DNAses, endonucleases, and exonucleases. Preferably, theplasminogen is suspended in an aqueous salt solution, including NaCl andNa₃PO₄, prior to freezing. The fibrin lysis reagent can also comprisePhospholipase A₂. Phospholipase A₂ is useful to help non-pathogen DNAdigestion by destroying phospholipid bilayers and, hence, destruction ofthe nuclear membrane.

In another embodiment, the enzymes of the fibrin lysis reagent can beprovided in a dried form and then when ready to be used in the presentmethods resuspended in a buffer solution using Potassium Phosphate as anaide to blood element solublization. It is imperative that thestreptokinase and plasminogen are not mixed with the buffer solutionuntil immediately prior to addition to the blood sample. The PotassiumPhosphate pH range can be about 7.8 to about 8.0, differentiated fromprior art claiming an effective pH range of 7.2 to 7.6. The prior artteaches the use of phosphate ion solutions with lower pH to act as atrue buffer; however, the method of the present invention allows foroptimal Phospholipase A₂ activity and Magnesium solubility. ThePotassium Phosphate acts as an essential component for blood matrixdisassembly when any of the enzyme combinations described herein areused. This contribution to blood matrix disassembly is comprised ofbiochemical interactions that are unrelated to buffering of pH. Thiscontribution of Potassium Phosphate to enzymatic driven blood matrixdisassembly has never been described before. When Potassium Phosphate isomitted and replaced with another buffer such as Tris-HCL, blood elementdisassembly does not occur and the blood sample matrix remainsincompatible for analysis. Magnesium can be present in the buffersolution as a divalent cation driving the activity of Phospholipase A₂in the presence of DNase. Prior art uses calcium as the classic divalentcation for driving Phospholipase A₂ activity; however, calcium is notcompatible with the phosphate ions essential for blood elementsolublization.

Enzymes that may be used in addition to or in place of plasminogenand/or streptokinase fall into five categories: 1) mutants or variantsof single chain urokinase type plasminogen activator; 2) mutants orvariants of tissue type plasminogen activator; 3) recombinant chimaericplasminogen activators; 4) conjugates of plasminogen activators andanti-fibrin monoclonal antibodies; and 5) compounds derived fromhaemophagous animals (including salivary plasminogen activator fromvampire bats), venom from southern copperhead snakes, antithrombolyticenzymes derived from leeches such as Hirudo medicincalis, Hirdinariamanillensis or Haementeria ghillanii, and staphylokinase from bacteria.

By including DNAse in the enzyme based Blood Processing Reagent, sampleprocessing is facilitated by the conversion of DNA of the patient'sblood cells into short fragments thereby contributing to a more rapidand efficient protein hydrolysis process during DNA extraction andlowering the burden of inhibitory DNA. Similarly, introduction of anendonuclease or an exonuclease produces a similar advantage. Theaddition of DNAse (a DNA nuclease), endonuclease, and/or exonuclease inthe methods of the invention provides for the conversion of DNA intoshort fragments. This conversion of DNA into short fragments contributesto a more rapid and efficient protein hydrolysis process during DNAextraction. This conversion of the patient's blood DNA into shortfragments is done while the bacterial DNA is protected. The shortfragment DNA is carried less efficiently through the DNA extractionprocess and hence represents a smaller proportion of total DNA product.As a result, the reduced patient DNA level presents less of aninhibitory component to the nucleic acid sequence based reactions. Whathuman DNA that does carry over into the sample extract is processed byDNAse, endonuclease, and/or exonuclease, preferably in the presence ofaurintricarboxylic acid. Other nuclease inhibitors that can be usedinclude salts of ATA, e.g., ATA triammonium salt, Ethyleneglycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid, Netropsindihydrochloride, or 1,10-Phenanthroline monohydrate,formaurin-dicarboxylic acid, Evans Blue (a structural analogue ofsuramin), vanadyl ribonucleoside complexes, nuclease inhibitors based onpoly vinylsulfonate, and the nuclease inhibition enhancer ZnCl₂ in sucha way that the human does not inhibit downstream nucleic acid baseddetection systems.

The use of nuclease inhibitors such as these, in reactions intended tohydrolyze DNA with nucleases, teaches against the art. The outcome ispreservation of pathogen DNA contained within intact pathogens whilepatient DNA is processed to facilitate proteinase K digestion plusoverall breakdown of the blood sample components when other reagentsspecified herein are used and also to process the patient DNA so that itwill not inhibit downstream nucleic acid detection reactions.

The enzyme based Blood Processing Reagent comprising plasminogen mayfurther comprise Phospholipase A₂, DNase, Endonuclease, Exonuclease,Lipase, plasminogen, streptokinase, staphylokinase, urokinase, plasmin,warfarin, monteplase, tenecteplase, reteplase, lanoteplase, pamiteplase,or any other modified tissue type-plasminogen activator,antithrombolytic enzymes derived from leeches such as Hirudomedicincalis, Hirdinaria manillensis or Haementeria ghillanii,plasminogen activators from the common vampire bat (Desmodus rotundus),mutants of plasminogen activators, chimeric plasminogen activators,conjugates of plasminogen activators, and any other plasminogenactivators from animal or bacterial origin, and combinations thereof.Dried lysis reagent may be suspended in pellets of trehalose buffer andpackaged into tubes as a dry reagent. The dried reagents may then beresuspended in a buffer, added to a 1 to 10m1 volume of blood andincubated for 5 to 20 minutes at room temperature. More specifically,the dried reagent can comprise 1,500 to 4,500 KU Phospholipase A2, 5,000to 10,000 U Streptokinase, 2 to 10 U Plasminogen, 200 to 3,650 U DNase,200 to 4,000 U Endonuclease, and 10,000 to 100,000 U Lipase, andoptionally one to fifty milligrams of purified (85 to 98% pure)staphylokinase, urokinase, plasmin, warfarin, monteplase, tenecteplase,reteplase, lanoteplase, pamiteplase, or any other modified tissuetype-plasminogen activator, antithrombolytic enzymes derived fromleeches such as Hirudo medicincalis, Hirdinaria manillensis orHaementeria ghillanii, plasminogen activators from the common vampirebat (Desmodus rotundus), mutants of plasminogen activators, chimericplasminogen activators, conjugates of plasminogen activators, and anyother plasminogen activators from animal or bacterial origin, andcombinations thereby.

One embodiment of the present invention includes concentrating andextracting analytes such as prions, toxins, metabolic markers, cancerousmatter, disease state markers, and/or pathogens such as bacteria, virus,and fungi from a volume of blood by introducing a Blood ProcessingReagent to expose analytes and/or pathogens in an aggregated bloodsample and analyzing the blood sample for the particles and/or pathogensnow readily identifiable following extraction from the aggregate. Theenzyme based Blood Processing Reagent may comprise plasminogen andstreptokinase. The plasminogen and streptokinase may be frozen incoincident relation until the fibrin lysis reagent is needed. Thestreptokinase then reacts with the plasminogen to form plasmin uponthawing. The plasminogen may be suspended in an aqueous salt solutionprior to freezing. Suitable salt solutions may include NaCl, NaPO₄ orthe like. Suitable detergents include methyl6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside, Triton X-100, and Saponin.To enhance analysis, nucleic acid from particles and/or pathogens may beamplified via polymerase chain reactions (PCR). As an alternative tofreezing, enzyme based Blood Processing Reagent may include driedstreptokinase and dried plasminogen as the fibrin lysis reagents. Thedried reagents may then be mixed and distributed into disposable testcontainers. This embodiment may be particularly useful for field-testingin locations where sophisticated laboratory equipment and controls areunavailable.

The enzyme based Blood Processing Reagent treated sample solution may becentrifuged for approximately 20 minutes at 5,000 to 5,500×g at atemperature of 10 to 20° C., the supernatant decanted, and the pelletwashed. The pellet may be washed three times with a 10 to 20 mM solutionof Ecotine/20 mM HEPES pH 7.7 and/or a 10 to 20 mM solution ofsucrose/20 mM HEPES pH 7.7 . The resultant sample may then be subjectedto nucleic acid extraction methods. Materials and methods for nucleicacid extraction are commercially available. The Blood Processing Reagentmay be used to treat whole blood samples for 10 minutes. The sample maythen be exposed to various embodiments of the Lyses Reagent or filteredwith a 0.22 to 0.45 μm Polyethersulfone (PES) filter unit, optionallywashed with 10 to 200 mM Aurintricarboxylic Acid, subjected again tolyses and nuclease inactivation using a solution comprising 12.5 to 25mg proteinase K, preferably 0.5-1.6% methyl6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside or less desirably 1 - 1.5%SDS (sodium dodecyl sulfate), and 10 to 20mM sodium citrate buffer pH7.8 to pH 8.4 may be utilized. Lysate may be eluted from the filtersurface by addition of 3.5 to 4.2 M guanidine isothiocyanate pH 6.4 andextracted according to various embodiments of prior art nucleic acidextraction known commonly as the “Boom” method. Optionally dried reagentmay be added.

Nuclease inhibitors that can be used in place of or in conjunction withATA in the materials and methods of the present invention includeEthylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic, Netropsindihydrochloridem 1,10-Phenanthroline monohydrate, formaurin-dicarboxylicacid, GR144053F, Evans Blue, vanadyl ribonucleoside complexes, andMelittin.

The pathogens, components, or other matter obtained from a sampleaccording to the present methods can be analyzed and identified usingany suitable means known in the art. For example, the solution obtainedfollowing the above steps may be applied directly to a biosensor devicewhich can capture and detect pathogenic or native disease state markersdeveloped by the animal against pathogens present in its blood.Alternatively, the solution may be applied directly to a liquidchromatography mass spectrometry device which can detect mass signaturesassociated with the structural components of the pathogens.

The enzyme based Blood Processing Reagent can comprise detergent andsalts. The enzyme based Blood Processing Reagent may aid blood elementsolublization by introducing 10 to 30 mM Potassium Phosphate at a pHrange of 7.8 to 8.0, driving Phospholipase A₂ activity by adding 10 to80 mM Magnesium Chloride as the divalent cation, adding 20 to 150 mMSodium Chloride, and including 10 to 200 mM Aurintricarboxylic Acidduring the DNase incubation process. The enzyme based Blood ProcessingReagent may also include 1.0 to 1.2% Triton X-100 or alternatively thereagents may include combining 20 to 35 mM methyl6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside and 0.05 to 0.1% Saponin or20 to 35 mM methyl 6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside byitself; and storing the enzymes by using a trehalose buffer. Storing theenzymes is accomplished by using a trehalose buffer in combination withmethyl 6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside. The trehalosestorage buffer comprises 10 mM Potassium Phosphate, 0.01 to 0.04% TritonX-100, 1 to 5 mM Dithiothreitol, and 0.3 to 0.5 M Trehalose.

In one embodiment, the sample Lyses Reagent used with the inventioncontains urea. The sample Lyses Reagent is preferably provided in adried form so as to minimize the downstream sample volumes and obviatethe procedure of having to prepare a proteinase K (PK) solution (since asolution comprising PK in 3.5 to 7.0 M urea is not stable for longperiods of time of time). The Lyses Reagent can also contain a detergentsuch as methyl 6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside. Thisdetergent can be dehydrated with the urea and proteinase K and providedin dry form. The detergent helps to disaggregate proteins but does notdenature them.

In another embodiment the nuclease present in the enzyme driven BloodProcessing Reagent can be used to nick human DNA while bacterial DNA isleft intact. The human DNA may then be removed after nucleic acidextraction by any method by using Single Strand Binding Proteinimmobilized on magnetic beads. PCR inhibition presented by the human DNAfound in large volume blood samples extracts can be reduced as evidencedby testing where said human DNA removal produced earlier PCR noise bandcrossing points and improved sensitivity compared to no human DNAremoval (Table 6).

In FIG. 1, a blood draw 30 is performed on a patient. A solution of PBS,pH 7.4 and 1.2% Triton X-100 is added, the blood is vortexed andcentrifuged 40 creating pellet 60 in a 15 ml tube 50. Preferably,resins, metal hydroxides, and/or nano materials may be added with thePBS/Triton X-100 solution to capture particles such as bacteria, virus,fungi, cancerous cells, prions, toxins and the like to contributegreater density to these particles. The increase in particle densityallows lower speeds to run during centrifugation.

The supernatant is decanted leaving a fibrin aggregate. A fibrin lysiscomponent 70 is added to tube 50 dissolving the fibrin aggregate andleaving pathogens 65 exposed for analysis. Pathogens 65 are vortexed,centrifuged, and subject to lysis to extract the pathogen DNA. The DNAis then replicated 90 and analyzed 100 for the identity of the suspectedpathogen.

In an alternative embodiment of the invention, a device would be used toobviate the need for a centrifuge. The device will use flexibleelectrodes similar to a fish gill to collect particles (such asbacteria, virus, cancerous cells, prions, or toxins). The electrodeswill also be used to collect resins and nano materials that have theseparticles attached to them. The device will resemble a bubble on asurface. An electrical potential will be used to accelerate pathogencapture. The device can be compressed to allow efficient removal of thecontents. The device would preferably have the following properties: (1)a rigid base layer and flexible top layer; (2) flexible gills to bemounted on either the top or bottom layer; (3) Strepavidin andhyaluronic acid strands functionalized with bioactive peptides,antibodies, aptomers, molecular imprinted polymers, or metals thatattract particles such as bacteria, virus, fungi, toxins, metabolicmarkers, disease state markers, or chemical agents are to be depositedon the flexible gill electrodes; (4) the flexible layer will haveelectrodes deposited on it; (5) counter electrodes for the gillelectrodes will reside on the opposite side; (6) the average dead volumeof the device is 300 micro liters—it is preferred that there is to be noresidual material in the device after squeezing out the material fromthe device; and (7) polyimide will form the flexible portion and theelectrodes will be made of Pt, Au, or carbon. The device is preferablyused as follows: (1) flow liquid into the device and apply voltage atthis time; (2) add chemicals and heat the device; and (3) squeeze outthe device to remove all contents. The device is used to prepare asample for analysis of particles (such as bacteria, virus, cancerouscells, prions, or toxins) using spectrophotometric, mass spectroscopy,antibodies, culture, or nucleic acid-based (e.g. PCR, NASBA, TMA)detection systems.

A filtering device may be used to filter out the particles from bloodtreated with the Triton X-100/PBS/magnesium solutions with enzymesselected from the group of streptokinase, plasminogen, phospholipase A₂,DNase, and lipase. A filtering device may also be used to filter out theparticles from blood treated with a combination of methyl6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside, Saponin, and PBS/magnesiumplus enzymes selected from the group of streptokinase, plasminogen,phospholipase A₂, DNase, and lipase. After washing away the enzyme anddetergent treatment reagents and any residual broken down bloodcomponents, the particle is ready for analysis or further processing.

The preparation of the fibrin lysis reagent is shown as Protocol 1 inFIG. 2 wherein NaCl, MnCl, DTT, DNAse, and plasminogen are added tomixing tube 110. Sodium phosphate is then added to mixing tube 110 andthe solution is distributed into 1.5 ml reagent tubes 120 placed on ice.The reagent tubes 120 are frozen to −75° C. for approximately 20minutes. Approximately 2,700 U of streptokinase 130 is added to the wallof reagent tubes 120 just above the frozen plasminogen solution.

Tables 1-4 provide PCR results derived from testing blood samples seededwith encapsulated vegetative avirulent Bacillus anthracis were grownaccording to CDC protocol # CDC.DFA.1.2, stored in 15% glycerol TSB, andfrozen at −75° C. Stocks of avirulent Yersinia pestis grown in TSB at37° C., frozen in 15% glycerol TSB, and frozen at -75° C. Bacterialcounts were tested at the time of harvest and retested at the time ofsample spike.

Figures for average Bacillus anthracis CFU per six ml of human blood arederived from post-freezing testing given the large standard deviationencountered in side-by-side post freezing dilution events. Nosignificant cellular death is recognized or expected. A 30% cellulardeath rate is the highest that is reasonably expected in the worstcircumstances. A conservative approach would be to increase allcalculated Bacillus anthracis CFU by 30%.

Figures for average Yersinia pestis CFU per six ml of blood are derivedfrom pre-freezing testing. The low standard deviation of pre-freezingcount replicates and concordance with post-freezing testing allows useof the pre-freezing bacteria count numbers. This is a conservativeapproach that can be utilized given the now predictable results that arederived from storing and diluting this organism.

The present invention reproducibly generates analyte DNA appropriate forPCR testing of pathogens, such as Bacillus anthracis, using patientblood samples that are up to 3 months old. Sensitivity is 100% at <10CFU/ml of human blood when using 6 ml of blood collected in a BectonDickinson Vacutainer (Tables 1 and 2). This protocol also allowsdetection of Yersinia pestis at 100% sensitivity at <10 CFU/ml for atleast one of four oligo sets according to the more limited data gatheredfor this organism (Table 4). It should be noted that CDC does notconsider samples positive for Y. pestis unless two oligo sets produce anacceptable PCR signal. In accordance with Protocol 1, FIG. 3 shows apreferred method of the setup of extraction reagents according to theinvention. FIGS. 4-5 show a method of bacterial recovery and fibrinlysis according to the invention. FIGS. 6-9 show a preferred method ofbacterial lysis and nucleic acid extraction according to the invention.

In an alternative embodiment, as shown in FIGS. 10-12b, the individualenzymes of streptokinase and plasminogen are made into dried powders,mixed, then distributed to disposable tubes. In another embodiment,Phospholipase A₂, plasminogen, DNase or Endonuclease, and lipase aresuspended and dried in pellets of trehalose buffer. AlthoughPhospholipase A₂ is preferred, any enzyme that will destroy nuclearmembrane while keeping bacterial cell wall or viral coats intact mayalso be used. Streptokinase is likewise suspended and dried in pelletsof trehalose buffer. At least one pellet of the plasminogen and onepellet of the streptokinase are packaged into tubes as dried reagents.

Dried reagents of the invention can be resuspended in a 10 ml buffersolution comprising 10 to −30 mM Potassium Phosphate, 10 to 80 mMMagnesium Chloride, 20 to 150 mM Sodium Chloride, 10 to 200 mMAurintricarboxylic Acid and 1.0 to 1.2% Triton X-100. AurintricarboxylicAcid is evidenced to provide a level of protection to bacterial nucleicacid without impeding human DNA digestion. The use of AurintricarboxylicAcid is not described in prior methods of human DNA digestion. Methyl6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside and/or Saponin can besubstituted for Triton X-100. In one embodiment, the methyl6-O—(N-heptylcarbomoyl)-α-D-glucopyranoside is used at 20 to 35 mM andthe saponin is used at 0.05 to 0.19 concentration. The methyl6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside is stored with thephospholipase A₂, plasminogen, DNase I, and lipase in a Trehalosestorage buffer. Substitution of the Triton X-100 with the methyl6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside and/or saponin solutionallows for the efficient activity of Phospholipase A₂, provides theaction of breaking up protein aggregates without denaturation, and ismore genial to bacterial walls than Triton X-100. Use of Saponin withmethyl 6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside in this combinationis not described in the prior art. The Trehalose storage buffer cancomprise 10 mM Potassium Phosphate pH 7.4, 0.01 to 0.04% Triton X-100, 1to 5 mM Dithiothreitol, and 0.3 to 0.5 Trehalose. The buffer and enzymemix are then immediately combined with a 10 ml blood sample, which maybe scaled down to 1 ml. The sample is then incubated at room temperaturefor 5 to 10 minutes. The aforementioned components aide blood elementsolublization through minimizing certain particulates that wouldotherwise clog filters, impair biosensors or mass spectrometry devices,and impede nucleic acid extraction. Solublization occurs while human DNAis processed and as viral and/or bacterial DNA remain intact.

In accordance with Protocol 2 and 4, a preferred enzyme based BloodProcessing Reagent combination is comprised of Streptokinase,Plasminogen, DNase or Endonuclease, Phospholipase A₂, and Lipase.Alternatively, an enzyme combination comprising Streptokinase,Plasminogen, DNase or Endonuclease, and Phospholipase A₂ may also beused. In another alternative combination, an enzyme combinationcomprising Streptokinase, Plasminogen, and DNase or Endonuclease may beused. Alternatively, an enzyme combination comprising DNase, and/orEndonuclease, and/or exonuclease, plus Phospholipase A₂ may be used.Alternatively, an enzyme combination comprising DNAse, and/orendonuclease, and/or exonuclease, plus Phospholipase A_(2,) plus Lipasemay be used. The biochemical impact on blood matrix disassemblyresulting from various combinations of Streptokinase, Plasminogen,DNase, and Phospholipase A₂ is described in FIG. 14.

As shown in FIG. 10 with Protocol 2, the sample is centrifuged for aperiod of 20 minutes at 5,000 to 5,500×g at a temperature between 10 to22° C. after incubation. The supernatant is then decanted and the pelletwashed three times with a 10 to 20 mM solution of Ecotine/20 mM HEPES pH7.7 and/or a 20 to 30 mM solution of Sucrose/20 mM HEPES pH 7.7. Thepellet is then heated to 90° C., centrifuged X 5 minutes at 13,00 RCF,and the supernatant is used for PCR analysis.

Alternatively after incubation, the Protocol 2 sample is centrifuged insimilar fashion and the supernatant decanted, followed by sample lysisand DNase or Endonuclease inactivation using 12.5 to 25 mg Proteinase K,1 to 1.5% Sodium Dodecyl Sulfate (SDS), 10 to 200 mM AurintricarboxylicAcid and 10-20 mM Sodium Citrate buffer pH 7.8 to pH 8.4. The sample isallowed to incubate at room temperature for 10 minutes. The digestedsample may then be applied to any commercially available nucleic acidextraction method, shown in FIG. 10B.

In yet another alternative, referred to as Protocol 3 and depicted inFIG. 11, the sample is filtered with a 0.22 to 0.45 μm filter unit andwashed with 10 to 20 ml of 10 to 200 mM Aurintricarboxylic Acid,followed by sample lysis and DNase or Endonuclease inactivation. Samplelysis and DNase or Endonuclease inactivation is accomplished by using12.5 to 25 mg Proteinase K, 1 to 1.5% SDS, 10 to 200 mMAurintricarboxylic acid, and 10 to 20 mM Sodium Citrate buffer. Thesample is then incubated at room temperature for 10 minutes. Addition of3.5 to 4.2 M Guanidine Isothiocyanate pH 6.4 is necessary to elute thelysate from the filter surface. The nucleic acid extract may then befurther purified using a commercially available method. Data derivedfrom this approach is contained in FIG. 13.

Another alternative, referred to as Protocol 4 and shown as FIG. 12A,the sample is applied directly to a biosensor device that will captureand detect bacteria, virus, fungi, toxins, prions, chemical agents,metabolic markers or native disease state markers developed by thepatient's own body in response to these pathogens and agents present inthe blood sample.

In yet another Protocol 4 alternative shown in FIG. 12B, the sample isapplied directly to a liquid chromatography mass spectrometry devicethat will detect mass signatures of structural components that comprisebacteria, virus, toxins, prions, and chemical agents present in theblood sample or native disease state markers developed by the patient'sown body in response to these pathogens and agents present in the bloodsample.

The subject invention also concerns a method for preventing ordecreasing inhibition of a nucleic acid based pathogen detection assayof a sample by host DNA, such as human DNA, despite the presence of hostDNA at concentrations in the sample that would normally inhibit theassay, said method comprising contacting said host DNA with a nucleaseand a nuclease inhibitor. Examples of such assays are described hereinand in U.S. patent application Ser. No. 10/604,779. In one embodiment,the nuclease is a DNAse, an endonuclease, or an exonuclease. Preferably,the nuclease inhibitor is ATA. Other nuclease inhibitors that can beused include Ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic,Netropsin dihydrochloride, 1,10-Phenanthroline monohydrate,formaurin-dicarboxylic acid, GR144053F, Evans Blue, vanadylribonucleoside complexes, and Melittin.

The subject invention also concerns materials and methods that can beused for the selective removal of ATA from a composition, such as thosecontaining nucleic acid. Typically, ATA is used in procedures forextracting and purifying RNA from cells, viruses, etc., because of itsactivity as a ribonuclease inhibitor. Using the claimed invention, thepotent ribonuclease inhibitor ATA will always be present during theportion of the nucleic acid extraction process where protein hydrolysisis allowed to proceed at optimal conditions (i.e., with ATA and notchaotrophic salts such as guanidine thiocyanate). The composition can beprovided in either solution form or dry, solid form. Preferably, thecompositions are provided in a dry solid form to which a liquid or fluidis subsequently added. In an exemplified embodiment, a composition ofthe invention is used in combination with the lysis reagents describedherein.

In one embodiment, a method of the invention comprises contacting asample that comprises ATA and, optionally, nucleic acid, with a ureacomposition optionally comprising DTPA (diethylenetriaminepentaacetate).In one embodiment, the sample can comprise any combination of reagentsas described in a Lyses Reagent of the invention. A urea/DTPAcomposition of the invention can be prepared by combining urea with DTPAand optionally EDTA, sodium citrate, and enough of a base, such assodium hydroxide to achieve pH 8.0 as defined in Table 5. In oneembodiment, the urea/DTPA mixture is heated to about 400 to 600° C. forabout 1 to 4 hours, dried, ground to a powder, and optionally combinedwith proteinase K and methyl 6-O—(N-heptylcarbamoyl)-α-D-glucopyranosideprior to addition to the sample in which ATA is to be removed. Theurea/DTPA reagent is preferably provided in a dried form so as tominimize the downstream sample volumes and obviate the procedure ofhaving to add proteinase K (PK) in a separate step (since PK is notstable for long periods of time in 6.0 to 7.5 M urea). The groundurea/DTPA reagent is dried under vacuum and added (at 360 mg reagent perml of blood sample) to blood treated with fibrin lysis reagentsdescribed herein. The sample treated with urea/DTPA is incubated 5 to 10minutes at about 65° C. When samples treated with ATA and a urea/DTPAreagent of the invention are combined with prior art nucleic acidextraction protocols where binding matrices such as silica or othermaterials that bind nucleic acids in the presence of chaotrophic saltsor where precipitation and centrifugation is used, the ATA will not copurify with the nucleic acid extract. In another embodiment, theproteinase K can be inactivated by exposure to temperatures above about80° C. for 5 to 10 or more minutes, the sample then cooled to belowabout 40° C., wherein urease is then added to about 1,000 to 100,000U/ml to break down the urea. The sample can then be applied directly toa nucleic acid array device.

Using the subject methods in conjunction with PCR, 10 CFU Bacillusanthracis per 10 ml of blood can be detected. Also, there was nodifference in the RT-PCR kinetics derived from PBS samples where 1 ng ofMS2 RNA was seeded into nucleic acid extracts made with and without ATA.Also, 1,000 pfu polio sabin III virus/8 ml SPS whole blood was detectedby RT-PCR when lysis reagents described herein were combined with theurea/DTPA reagent and protocol described above. By using a urea/DTPAreagent of the invention, ATA that was present prior to the proteinase Kdigestion step did not have a negative impact on the PCR kinetics usingthe nucleic acid extracts that were prepared using the subject methods.

In another embodiment, if a sample is not processed with Lyses Reagent,such as those described herein, then a buffer comprising only ATA can beadded to the cells as a first step and subsequently treated as outlinedabove.

In another embodiment, urea can be added to about 6.0 to 7.5 M to an ATAcontaining sample, and then combined with prior art chaotrophic saltbased binding buffers and silica binding matrices, conduct the protocolaccording to the literature citation or manufacturer specifications withthe exception of heating the chaotrophic salt based binding and washbuffer to about 55 to 65° C. prior to use with the sample. The reactionof urea with the ATA plus the combination of this solution withchaotrophic salt at 55 to 65° C. followed by application to a silicabased nucleic acid capture matrix allows the selective binding ofnucleic acid to the matrix and exclusion of ATA (which is passed out inthe capture matrix flow through). It is the combination of reaction withurea and heat that provides for the exclusion of ATA from the silicacapture matrix while nucleic acid binds readily. The above describedurea/DTPA reagent produced by heating the urea and DTPA combination tobetween 400 to 600° C. during production eliminates the need for thischaotrophic salt heat step and allows for more complete removal of theATA.

In another embodiment, blood samples can be treated with ATA containingmixtures described in combination with pathogen capture using bioactivepeptides functionalized on hyaluronic acid where the hyaluronic acid inturn acts as a polymeric waveguide. The hyaluronic acid is labeled withbiotin via carboxyl groups or amines and the biotin is subsequentlyremoved via dialysis. Strepavidin is cross-linked and the cross-linkeris removed via dialysis. The cross-linked strepavidin is added in 100 to10,000 molar excess to the biotinylated hyaluronic acid and incubatedabout 4 to 10 hours with or without an applied electrophoretic ordielectrophoretic field. Alternatively, the strepavidin is added in thedescribed ratios, incubated for about 1 to 4 hours with mixing, combinedwith a photo- activated cross-linking reagent, and cross-linked withinas lithography system in order to generate structures positioned withina sample flow path. In this system a calcium release at the site ofpathogen capture via bioactive peptide or annealing of RNA species isused to trigger the local conversion of reporter molecule labeledfibrinogen to an insoluble fibrin aggregate at the site of pathogencapture via bioactive peptide or annealing of RNA species upon thematrix of the hyaluronic acid polymeric waveguide. As used herein,bioactive peptides include native and modified non-specific virusbinding peptides most optimally, such as lactoferrin or fatty acidmodified lactoferrin, and native and modified non-specific bacteriabinding peptides, most optimally, such as Cecropin P1, but alsoincluding, for example, protamine, Buforin I, Buforin II, Defensin,D-Magainin II, Cecrpin A, Cecropin B, Lectin PA-1, and Tritrpticin. Themodified peptides may be altered in terms of amino acid content andinclude the salts, esters, amides, and acylated forms thereof.

In another embodiment, the bioactive peptides functionalized upon thehyaluronic acid (that is cross linked via biotin and strepavidin) act aspathogen capture moieties. Upon pathogen or biomarker capture, thehyaluronic acid is broken down using 1,000 to 1,000,000 units ofhyaluronidase/ml of sample within the device.

In another embodiment, the ATA, magnesium chloride, and potassiumphosphate components described in the lysis buffer of the presentinvention are combined, brought to about pH 9.2 to pH 10 in batches of100 ml, and heated to boiling until a dry residue forms. The dry residueis ground up, dried further under vacuum, and added to the other enzyme,detergent, and Trehalose components, such as those described herein. Inthis way the blood can be added directly to dried pellets of Trehalosestabilized reagent where no other liquid or dry components are added forinitial blood pretreatment. The dried urea and proteinase K reagent isthen added followed by processing as described herein. When this driedreagent system was independently evaluated by government scientists 10CFU Yersinia pestis/ml whole blood was detected via PCR.

The subject invention also concerns compositions comprising ATA that canbe used to isolate nucleic acid from a sample. The composition can beprovided in either solution form or dry, solid form. Preferably, thecompositions are provided in a dry solid form to which a liquid or fluidis subsequently added. B. anthracis seeded 8 ml blood sample extractswere tested in the “canonical” SNP (canSNP) analysis system. The 250CFU/8 ml blood sample gave a strong signal and was easily typed by theKeim Genetics Lab personnel (FIG. 16). Antibody capture of Salmonellafrom whole blood was conducted, followed by PCR and no difference wasfound compared to capture, wash, and extraction from a PBS/Plasmamixture (Table 7). The CDC RRAT Bioterrorism Lab run by Dr. Rich Meyerhas detected 100 CFU Brucella abortis/8 ml whole blood using the enzymebased Blood Processing Reagent. USAMRIID Diagnostics Divisioninvestigators have detected 10 CFU Yersinia pestis/1 ml whole bloodusing the enzyme based Blood Processing Reagent. The biosensor group atUniversity of Texas at Austin detected 1 ng C-Reactive protein / mlwhole blood in a biosensor that would normally never accept a wholeblood sample (FIG. 17).

The subject invention also pertains to materials and methods forefficiently removing patient or host (e.g., human) DNA that has beenprocessed by DNAse, endonuclease, or exonuclease while pathogen DNAremains inside intact pathogens. E. coli SSB proteins are known in theart and can be used in the methods of the invention. In one embodiment,the SSB is immobilized on magnetic beads. In an exemplified embodiment,the SSB is biotinylated and the solid matrix has avidin or streptavidinattached to the surface, and the SSB is bound to the matrix via thebiotin-avidin binding. The sample is then circulated for several minutesat about 37° C. The sample containing the nucleic acid and SSB-matrix isthen washed one or more times with a suitable wash buffer, such as 2 mMTris (pH 8.0). The sample and SSB-matrix is then heated to 90 to 100° C.for a few minutes, the SSB-matrix is separated from the sample and thesample collected. The sample contains purified nucleic acid and can bestored, purified further, or used in PCR, etc.

In one embodiment, a method of the invention comprises contacting asample that comprises nucleic acid and ATA with a nucleic acid bindingmatrix. The binding matrix is subsequently contacted with a bindingbuffer solution comprising a thiocyanate, such as guanidine thiocyanate.After contact with the binding buffer, the binding buffer and sample areremoved from contact with the binding matrix, preferably by evacuationof the buffer and sample away from the binding matrix. The bindingmatrix can optionally be washed one or more times with wash buffer. Inone embodiment, blood is collected in a tube containing any commerciallyavailable anticoagulant with inversion of the tube 8× immediately postcollection. Store the blood at room temperature and process within 14days post collection. Fresh blood (0-48 hours old) may be best toexamine. This is the optimal condition. Refrigerated blood that is 3months old will also work for instance. Frozen blood may not work ifbacteria have lysed via crystallization. Add one volume of whole bloodto one tube of Blood Processing Reagent powder (160 mg powder/ml wholeblood), vortex on high X 15 seconds, and incubate X 8 min @ roomtemperature. Dump one tube of Lysis Reagent powder into the blood sample(350 mg powder/ml whole blood), vortex on high for 15 seconds andincubate in a 65° C. water bath X 10 min. Add one volume (equal tovolume of blood) of 4.1 M Guanidine Thiocyanate, 9.5% Triton X, 200mMTris HCL, pH 6.0 and vortex X 5 sec. Contact this mixture with a silicamatrix such as a commercially available spin column or magneticparticles coated with silica, or other materials designed to bindnucleic acids in the presence of a chaotrophic salt. Wash the silicamatrix with a solution of 4.1 M Guanidine Hydrochloride, 50 mM Tris HCL,pH 6.4. Follow this with a wash using 70% ethanol. Elute the nucleicacid using 2 mM Tris-HCL pH 8.5. The material is now ready for storageor PCR testing.

The examples described herein illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

TABLE 1 Bacillus anthracis Blood Protocol Data Set pXO2 Primer/ GenomicPrimer/ Ave. Comments on Probes - Crossing Probes - Crossing CalculatedSample Type All Point on Light Point on Light CFU/6 ml Samples Tested 2Sample Number Cycler Cycler of blood Days Post Spiking M3200253BA1 36.7537.76 13.75 Spiked Positive M3200253BA2 36.59 37.86 13.75 SpikedPositive M3200253BA3 35.97 38.10 13.75 Spiked Positive M3200253BA4 37.2639.53 13.75 Spiked Positive M3200253BA5 35.36 40.11 13.75 SpikedPositive M3200253BA6 36.35 45.19 13.75 Spiked Positive M3200253BA7 36.6238.64 13.75 Spiked Positive M3200253BA8 37.04 39.51 13.75 SpikedPositive M320020BA9 0.00 0.00 0.00 Blank M/3200226BA1 37.16 39.35 1.38Spiked Positive M/3200226BA2 36.79 40.28 1.38 Spiked PositiveM/3200226BA3 37.92 39.94 1.38 Spiked Positive M/3200226BA4 37.49 40.161.38 Spiked Positive M/3200226BA5 39.66 40.26 1.38 Spiked PositiveM/3200226BA6 39.31 41.19 1.38 Spiked Positive M/3200226BA7 38.48 40.731.38 Spiked Positive M/320020BA8 0.00 0.00 0.00 Blank

TABLE 2 Bacillus anthracis Blood Protocol Data Set: Comparison of Bloodfrom Two Different Individuals and Evaluation of Blood Sample Age pXO2Primer/ Genomic Primer/ Ave. Comments on Probes - Crossing Probes -Crossing Calculated Sample Type All Point on Light Point on Light CFU/6ml Samples Extracted Sample Number Cycler Cycler of blood 84 Days PostSpiking V210253BA1 37.73 39.81 10.5 Blood Donor #1 V210253BA2 36.7439.05 10.5 Blood Donor #1 V210253BA3 36.51 37.99 10.5 Blood Donor #1V210253BA4 38.12 39.79 10.5 Blood Donor #1 V21020BA5 0.00 0.00 0.00Blank M210253BA1 37.86 39.81 2.25 Blood Donor #2 M210253BA2 37.84 39.222.25 Blood Donor #2 M210253BA3 37.24 38.52 2.25 Blood Donor #2M210253BA4 38.68 39.33 2.25 Blood Donor #2 M21020BA5 0.00 0.00 0.00Blank

TABLE 3 Bacillus anthracis Blood Protocol Data Set: Evaluation of BloodProtocol by a Department of Health Laboratorian Comments on pXO2 Primer/Genomic Primer/ Ave. Sample Type: All Probes - Crossing Probes -Crossing Calculated Blood Samples Point on Light Point on Light CFU/6 mlSame Batch as Sample Number Cycler Cycler of blood in Table 1M3200256BA1L 38.81 39.93 13.75 Spiked Positive M3200256BA2L 36.10 39.2613.75 Spiked Positive M/3200223BA3L 36.77 38.58 1.38 Spiked PositiveM320020BA4L 0.00 0.00 0.00 Blank

TABLE 4 Yersinia pestis Blood Protocol Data Set YP 2 YP 9 YP 12 YP 16Primer/ Primer/ Primer/ Primer/ Comments on Probes - Probes - Probes -Probes - Sample Type Crossing Crossing Crossing Crossing Ave. AllSamples Point on Point on Point on Point on Calculated Extracted 2 LightLight Light Light CFU/6 ml Days Post Sample Number Cycler Cycler CyclerCycler of blood Spiking M3180251EYP1 0.00 0.00 0.00 37.97 12.0 SpikedPositive M3180251EYP2 0.00 47.01 0.00 0.00 12.0 Spiked PositiveM3180251EYP3 41.56 0.00 0.00 40.29 12.0 Spiked Positive M3180225EYP40.00 0.00 0.00 38.98 24.0 Spiked Positive M3180225EYP6 40.20 44.01 39.6637.60 24.0 Spiked Positive M3180251FYP7 0.00 46.15 0.00 39.79 48.0Spiked Positive M3180251FYP8 40.48 43.59 41.70 35.47 48.0 SpikedPositive M3180251FYP9 40.20 41.88 38.67 34.23 48.0 Spiked PositiveM318020YP10 0.00 0.00 0.00 0.00 0.00 Blank

TABLE 5 Contents of powdered Urea/DTPA reagent (upon addition of 1 mlsample to 360 mg reagent Urea 6.0-7.5M Methyl6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside 10 to 20 mg/ml Proteinase K600-1,000 μg/ml EDTA 20-70 mM DTPA 20-70 mM Sodium Citrate 120 mM SodiumHydroxide add to pH 8.0

TABLE 6 Assay Reagents Provided by the CDC RRAT Bioterrorism LaboratoryCFU/6 ml Sample No HDR With HDR Blood Number Treatment Treatment 20 10.00 0.00 20 2 0.00 0.00 50 3 42.11 36.68 50 4 0.00 0.00 100 5 40.1936.88 100 6 0.00 39.24 200 7 44.17 38.58 200 8 0.00 36.91 400 9 40.4534.48 400 10 43.29 35.67

TABLE 7 Antibody Capture of Salmonella from whole blood treated withBlood Processing Reagent; PCR noise band crossing points. Avg. NoiseBand Avg. Noise Band CFU Salmonella/ Crossing Point Crossing Point 8 mlSample PBS/Plasma Treated Whole Blood 1,000 31.7 31.8 500 32.8 32.5 10033.5 33.8

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

We claim:
 1. A container comprising a biological sample with addedproteins prepared by adding to a biological sample: (a) 2 to 10 U of anisolated plasminogen; (b) 5,000 to 10,000 U of an isolatedstreptokinase; (c) 200 to 3,650 U of an isolated DNase; (d) 1,500 to4,500 KU of a phospholipase A₂; and (e) a detergent.
 2. The container ofclaim 1, wherein the biological sample comprises a bacterium.
 3. Thecontainer of claim 1, wherein the biological sample comprises a fungus.4. The container of claim 1, wherein the isolated DNase is an isolatedendonuclease.
 5. The container of claim 1, wherein the biological sampleis further prepared by adding to the biological sample 10,000 to 100,000U of an isolated lipase.
 6. The container of claim 1, wherein thebiological sample is blood.
 7. The container of claim 1, wherein thebiological sample is a fibrin aggregate.
 8. A reagent composition,comprising: (a) 2 to 10 U of an isolated plasminogen; (b) 5,000 to10,000 U of an isolated streptokinase; (c) 200 to 3,650 U of an isolatedDNase; (d) 1,500 to 4,500 KU of a phospholipase A₂; and (e) a detergent.9. The reagent composition of claim 8, wherein the isolated DNase is anisolated endonuclease.
 10. The reagent composition of claim 8, whereinthe composition further comprises 10,000 to 100,000 U of an isolatedlipase.